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The number of recognition events dropped significantly, to 20% of the unblocked conditions

The number of recognition events dropped significantly, to 20% of the unblocked conditions. thioglucose (coupled via acrylamide to a long PEG) but decreased for aminophlorizin binding. The former indicates that in the membrane-bound SGLT1 the pathway to sugar translocation involves several steps with different temperature sensitivity. The latter suggests that also the aglucon binding sites for transport inhibitors have specific, temperature-sensitive conformations. sodium/galactose symporter (vSGLT) (13) in the sodium- and galactose-bound state. Overall, a group of seven central helices contributes side-chain interactions for ligand selectivity. These are stabilized by seven supporting helices. The model proposed Ptgfrn recently by Sala-Rabanal (15) integrates the kinetic and structural data available to date into a six-step alternating access model. Our group has successfully used atomic force microscopy (AFM) and single molecule recognition force spectroscopy (16,C18) to probe the transporter in its natural environment embedded in the plasma membrane of living cells under near-physiological conditions (19). The extracellular location and accessibility of three extramembraneous loops (loop 6C7, loop 8C9, and loop 13C14) was identified. They form a vestibule for the entry of the sugar into the translocation pathway and contain the first of several sugar recognition sites. This vestibule is accessible to the sugar only in the presence of sodium (20, 21). Phlorizin acts as a competitive inhibitor of SGLT1 with an apparent of 1 1 m LY 344864 (22). The phlorizin carrier complex represents a dead end conformation of the transporter in which it is locked into a condensed, rigid conformation unable to mediate translocation (23, 24). Phlorizin consists of a pyranose ring (sugar residue) and two aromatic rings joined by an alkyl spacer (the aglucon moiety, phloretin) (22). It is supposed that phlorizin binds via a two-step mechanism to the sugar translocation site and an aglucon binding site of the transporter (8, 25). One of the extracellular loops, loop 13C14, was found to provide an additional aglucon binding site. Alkyl-glucosides, such as hexyl-glucoside, also inhibit glucose transport competitively with a of 10 m (26, 27). The sites of interaction between the aglucon of the inhibitors and loop 13C14 LY 344864 differ and overlap only partly (10). In the present work AFM was employed to further characterize the molecular interaction between SGLT1 and d-glucose and inhibitors with regard to their dynamics and forces. Molecular LY 344864 interaction between receptors and ligands is controlled by a complex array of intermolecular forces that can be characterized by their free energy landscape. AFM can be used to directly quantify the range and magnitude of the interaction forces between proteins and other molecules (28, 29). Dynamic aspects of bond rupture, dissociation rate constants, commonly used to describe the affinity between a ligand and a protein, and width of energy barrier, interpreted as LY 344864 the distance of the energy barrier from the energy minimum along the direction of the applied force, can be obtained by varying the loading rate of the force appliance. This provides insights into the molecular dynamics and the energy landscape for substrate/inhibitor-transporter complexes. Location of energy barriers and nature of interaction forces have been studied extensively for proteins by investigating their properties at different temperatures (30). We used a similar approach as it has been shown that sodium-dependent glucose transport is strongly temperature-dependent (11), ceasing below the transition temperature of the membrane lipids (31). In contrast, sodium-dependent, glucose-inhibitable binding of phlorizin is still demonstrable at temperatures close to 0 C.4 Therefore, studies were performed at 10, 25, and 37 C to investigate further the properties of the glucose translocation pathway and the inhibitor binding sites. EXPERIMENTAL PROCEDURES AFM Tip Functionalization 1-Thio–d-glucose (thio-glc) (Sigma), 2-aminoethyl -d-glucopyranoside, and 3-aminophlorizin were coupled to the AFM tip using a well established three-step.